Epigenetic modifications, such as DNA methylation and histone acetylation/methylation, play a critical role in dictating the function and behavior of an organism. Efforts to unravel the workings of the epigenome, however, are constrained by severe technical limitations in our ability to manipulate chromatin modifications. Existing approaches typically involve pharmacological agents or genetic modification and tend to have pleiotropic effects. This proposal aims to develop a universal set of tools for manipulating epigenetic modifications capable of targeting specific chromatin modifiers to any desired locus and precisely controlling the timing and magnitude of these modifications. The proposed platform is built on our recently demonstrated CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference system-a highly efficient RNA- guided genome-targeting system derived from the CRISPR bacterial immune system. The method requires only a single protein, dCas9, a catalytically inactive variant of the CRISPR Cas9 proteins, and a designed small guide RNA (sgRNA) with a region that is complementary to the gene of interest. The sgRNA guides binding of the dCas9-sgRNA complex to the complementary genomic site. We will develop a CRISPR RNA-based toolset for targeted and inducible manipulation of the epigenetic modifications First, we will develop a series of optimized dCas9-sgRNA pairs in which the DNA location of targeting is determined solely by the sequence of the sgRNA and the epigenetic impact of targeting is dictated by the effector domain that is recruited by dCas9. This will then be coupled with a genome-wide library of sgRNAs capable of targeting the dCas9 at will to roughly 100,000 positions in the human genome. Second, to enable inducible epigenetic controls, we will fuse the dCas9 proteins to a complement of diverse epigenetic modifier domains and effector domains that can be controlled by light or drugs. The epigenetic modifier domains will contain different catalytic and/or regulatory domains derived from natural epigenetic regulators, including both writer and eraser modules. Optogenetically and chemically gated interaction modules will be used to achieve spatial and temporal control. Third, we will apply these tools to a small set of pilot mechanistic and bioengineering studies to test how the CRISPR epigenetic toolbox can be utilized. Specifically, we will modify the epigenetic marks of the murine Oct4 and Sox2 loci as the endogenous experimental testbed, and study the importance of modifier recruitment positioning and dynamics in controlling chromatin state. Finally, we will develop programmable CRISPR insulators to control long-range chromatin interactions of human beta globin expression. Together, this project will provide a universal RNA-guided platform for precise spatial and temporal regulation of the epigenome. We envision this platform will be a critical tool for studying the interplay between multiple epigenetic modifirs at different loci, understanding the relationship between epigenomic programming and disease, and facilitate the development of therapeutic methods to rewrite and reprogram epigenetic marks.